The peroxisome is a ubiquitous organelle of eukaryotic cells. Peroxisome participate in a wide variety of metabolic processes and defects in peroxisome biogenesis are the cause of numerous human diseases. These lethal inherited disorders are caused by defects in the biogenesis of peroxisome membranes or peroximal matrix protein import but all patients display mislocalization of matrix enzymes and loss of virtually all peroxisomal metabolic functions. This proposal is designed to improve our understanding of these diseases by testing our current working model of peroxisomal matrix protein import. In this model, predominantly cytoplasmic, partially peroxisomal import receptors recognize newly synthesized matrix enzymes. Docking factors on the peroxisome surface recruit these receptors and, if the receptors contain newly synthesized matrix enzymes, they are transferred to a translocation apparatus. At this point, the enzymes are dissociated from their receptors and translocated into the peroxisome lumen. This model also predicts that a recycling event releases the import receptors to the cytoplasms for additional rounds of protein import. Specific Aim 1 examines the structure and function of the import receptors. Specific aim 2 explores the mechanisms of import that occur following recognition but prior to translocation. Specific aim 3 investigates several peroxin- peroxin interactions that are critical for matrix protein import. The fourth aim supports studies of peroxisomal enzyme translocation in vitro. The significance of these studies is that they will improve our understanding of eukaryotic cell biology and the molecular basis of human disease by testing our current hypothesis of peroxisomal matrix protein import.